GATA-1 is an essential hematopoietic transcription factor that coordinates proliferation arrest with cellular maturation, a function of relevance to normal tissue formation and cancer. Germline mutations in GATA-1 cause inherited dyserythropoietic anemia and thrombocytopenia, while somatic mutations are associated with acute megakaryocytic leukemia (AMKL). The extent of GATA-1 actions and its critical targets are not fully defined; here we propose to investigate its antiproliferative functions. Preliminary findings support the hypothesis that GATA-1 inhibits cell cycle progression by repressing mitogenic genes and activating antiproliferative ones. Remarkably, at least two oncogenes, Kit and Myc, are inhibited directly by GATA-1 in erythroid cells, highlighting a significant role for gene repression in the GATA-1 antiproliferative program. [unreadable] GATA-1 also triggers proliferation arrest and maturation of cultured GATA-1- megakaryocytes through pathways that are currently unexplored. Our current goals are to investigate the mechanisms by which GATA-1 regulates Myc, Kit (Aim 1), and four other potentially key cell cycle effector genes whose expression are controlled by GATA-1 (Aim 2), as identified in our preliminary studies. In Aim 2, we will also examine the functions of these genes by manipulating their expression in erythroid cells and determining the effects on the GATA-1 antiproliferative program. These experiments address the poorly understood role of GATA-1 in gene repression and characterize new molecular targets, several of which were not previously implicated in hematopoiesis. To complement our work on erythroid cells, we will investigate the effects of GATA-1 on megakaryocyte proliferation (Aim 3). Specifically, we will define the transcriptional program associated with proliferation arrest caused by normal GATA-1 and examine mutations previously associated with hyperproliferation and/or maturation defects. These findings will provide further insights into how altered GATA function causes AMKL. Moreover, comparative analysis between erythroblasts and megakaryocytes should reveal aspects of GATA-1-mediated cell cycle that are either common or unique to these related lineages. Our major long-term goals are to define the regulatory hierarchies through which GATA-1 orchestrates cell cycle arrest during normal hematopoietic differentiation and to apply this knowledge to the study of cytopenias and leukemia(s) associated with altered GATA-1 function [unreadable] [unreadable]